1. Field of the Invention
The invention provides a method and apparatus for preparing a suspension of single cells. The invention is characterized by the step of subdividing an animal or vegetable tissue sample by hydraulically forcing the sample back and forth through a finely apertured wall.
2. Description of the Prior Art
Recent techniques for study of animal and plant tissues have involved the culturing of independent cells in vitro and the proliferation of animal cells in vivo as by injecting, for instance, tumor cells into a laboratory animal. In vitro culturing techniques are especially useful when it is desired to study human tumor cells or to biosynthesize a desired component of a tissue. Both in vitro culturing techniques and in vivo proliferation techniques require mincing of the tissue sample, which may be a solid tumor, and preparing a single cell suspension from the minced sample. Similarly, biological and biochemical tissue assay techniques often require the preparation of a single cell suspension from the tissue sample.
Prior art methods for preparing suspensions of single cells are often unduly time consuming and frequently damage the cells. Such methods involve both chemical, i.e., enzymatic, and physical techniques. Enzymatic techniques involve treating a coarsely subdivided tissue sample with a suitable enzyme, such as trypsin, capable of digesting the cell clusters or clumps sufficiently to break down the clusters or clumps into single independent cells. However, it is believed that enzymes adversely affect at least the surface of the cell and, therefore, such enzymatically-treated cells and their cultured progeny may have characteristics which are different from those of the original sample. If enzymatically-digested tumor cells are cultured in vitro and an effort is then made to study the response of the tumor cells to drugs, the response of the cultured cells and their progeny to drugs may be different from that which would be exhibited by the same tumor cells cultured without a history of enzymatic treatment.
Mechanical techniques for preparing single cell suspensions suffer similar drawbacks. One method involves forcing the minced animal tissue sample through successively smaller gauges of hypodermic needles, using a syringe. However, the amount of force necessary to push the minced tissue through the single extended aperture of the hypodermic needle requires that the tissue sample be exposed to hydrostatic pressures so high as to result in possible injury to the cells. Moreover, the procedure is unduly slow and requires excessive amounts of laboratory personnel time.
Another prior art procedure involves placing the minced tumor sample upon the surface of a fine wire mesh screen and forcing the sample through the screen with, e.g., a rubber plunger. The subdivided sample so produced is recovered and the operation is repeated several times with increasingly finer mesh screens until the tissue has been sufficiently subdivided. This procedure is extremely time consuming. Moreover, the bottom of the screen is often placed upon a solid surface for support while the material is forced through the screen so that the cells are pressurized between hard surfaces on both sides of the screen with likelihood of excessive cell trauma.